Nucleic acid derivatives

ABSTRACT

This invention relates to a methodology for assessing the sensitivity of an HIV-1 sample to zidovudine and to diagnostic assays for use in such assessment.

This is a continuation of application Ser. No. 07/564,062, filed Aug. 8,1990, now abandoned.The present invention is a continuation of Ser. No.08/839,010, filed Apr. 23, 1997, now RE 37,918 E, which is a reissue ofSer. No. 07/984,255, filed Dec. 1, 1992, which is a continuation of Ser.No. 07/564,062, filed Aug. 8, 1990, now abandoned, which claims benefitunder 35 USC 119 from GB 8918226, filed Sep. 8, 1989.

The present invention relates to a method for assessing the sensitivityof an HIV-1 sample to zidovudine, and to diagnostic assays for use insuch assessment.

One group of viruses which has recently assumed a particular importanceare the retroviruses. Retroviruses form a sub-group of RNA viruseswhich, in order to replicate, must first ‘reverse transcribe’ the RNA oftheir genome into DNA (‘transcription’ conventionally describes thesynthesis of RNA from DNA). Once in the form of DNA, the viral genome isincorporated into the host cell genome, allowing it to take fulladvantage of the host cell's transcription/translation machinery for thepurposes of replication. Once incorporated, the viral DNA is virtuallyindistinguishable from the host's DNA and, in this state, the virus maypersist for as long as the cell lives. As it is believed to beinvulnerable to attack in this form, any treatment must be directed atanother stage of the virus life cycle and would, have to be continueduntil all virus-infected cells have died.

A species of retrovirus has also been reproducibly isolated frompatients with AIDS and is now named as human immunodeficiency virus(HIV-1) and is also known as human T-cell lymphotropic virus III (HTLVIII), AIDS associated retrovirus (ARV), or lymphadenopathy associatedvirus (LAV).

This virus has been shown preferentially to infect and destroy T-cellsbearing the OKT⁴ surface marker and is now generally accepted as theactiologic agent of AIDS. The patient progressively loses his set ofT-cells, upsetting the overall balance of the immune system, reducinghis ability to combat other infections and predisposing him toopportunistic infections which frequently prove fatal. Thus, the usualcause of death in AIDS victims is by opportunistic infection, such aspneumonia or virally induced cancers, and not as a direct result of HIVinfections.

The complete nucleotide sequence of the AIDS virus HIV-1 or as it waspreviously known HTLV-III has been elucidated (Ratner, L., et al.Nature, Vol. 313, p 277, 24 Jan. 1985).

Recently, HIV-I has also been recovered from other tissue types,including B-cells expressing the T⁴ marker, macrophages and non-bloodassociated tissue in the central nervous system. This infection of thecentral nervous system has been discovered in patients expressingclassical AIDS symptoms and is associated with progressivedemyelination, leading to wasting and such symptoms as encephalopathy,progressive dysarthria, ataxia and disorientation. Further conditionsassociated with HIV infection are the asymptomatic carrier state,progressive generalised lymphadenopathy (PGL) and AIDS-related complex(ARC). HIV-1 can also be present in other tissues or physiologicalfluids such as urine, plasma, blood, serum, semen, tears, saliva orcerebrospinal fluid.

3′-Azido-3′-deoxythymidine-(hereafter called “zidovudine”) is used inthe control of HIV infections including AIDS and ARC. Zidovudine is athymidine analogue whose triphosphate form inhibits the replication ofthe human immunodeficiency virus (HIV) by competitive binding to viralreverse transcriptase (RT) and DNA chain termination afterphosphorylation by cellular enzymes (Furman, P. A et al., Proc. Natl.Acad. Sci. USA, 1986, 83: 8333). It is an effective antiviral agent bothin vitro and in vivo against a variety of retroviruses (Mitsuya H etal., Cancer Res., 1987, 47: 2190; Ruprecht R. M et al., Nature, 1986,323: 476.) and has been demonstrated to improve the quality and lengthof life of patients with AIDS and advanced ARC (Fischl M. A. et al., N.Engl. J. Med., 1987, 317: 185; Schmitt F. A. et al, N. Engl. J. Med1988, 319: 1573; Greagh-Kirk T et al, J. Am. Med. Assoc., 1988, 260.:3009) and also in asymptomatics with low CD₄ ⁺ cell levels.

As with any anti-infective agent, concern about the potentialdevelopment of resistance has engendered extensive investigationsevaluating factors which might potentially alter the sensitivity ofretroviruses to zidovudine.

A study carried out to measure zidovudine sensitivity of HIV isolatesfrom patients with AIDS or ARC after zidovudine treatment has in factrevealed that a number of isolates from patients treated for six monthsor more showed reduced sensitivity to zidovudine whereas isolates fromuntreated individuals and those treated for less than six months showeduniform sensitivity to the drug (Larder, B. A., Darby, G, and Richman,D. D., Science, Vol. 243, 1731, 31st Mar. 1989).

At present the way to determine the sensitivity of HIV-1 strains tozidovudine is to isolate HIV-1 from a patient's peripheral bloodlymphocytes. HIV-1 isolates can be made by co-cultivation of peripheralblood lymphocytes (PBL's) with cells of the continuous line MT-2.(Harada, S., Koyanagt, Y., Yamamoto, N., Science 229, 563 (1985). Thisprocedure can take anything from between four and fourteen days beforeHIV-1 can be isolated. The diagnosis of resistant strains of HIV-1relies firstly on the isolation of virus and then on sensitivity testingby a tissue culture method and so is consequently extremely slow.

The present inventors have discovered the basis for resistance of HIV-1to zidovudine at the nucleic acid level. Five nucleotide substitutionshave been identified in the HIV-1 genome which result in a change offour amino acids in the RT protein. This discovery has importantimplications for the detection of resistant HIV isolates because of thehighly conserved nature of these nucleotide mutations in RT conferringresistance to zidovudine, and opens the way to the routine detection ofsuch resistant isolates.

It is possible to carry out a diagnostic assay for the screening ofbodily samples from patients for an assessment of the sensitivity ofHIV-I to zidovudine. Using the knowledge of the mutations identified asimportant in the development of highly resistant strains of HIV-1 suchan assay can be developed.

An analysis of a group of resistant mutants was carried out bynucleotide sequencing which allowed the identification of mutations inthe HIV RT gene that confer resistance to zidovudine. The complete RTcoding region (1.7 kb) was obtained for each isolate using polymerasechain reaction (PCR) amplification of infected cell DNA. The nucleotidechanges at the five positions in HIV-1 RT that confer resistance tozidovudine are illustrated in FIG. 1. Numbering of the nucleotides ofthe HIV-1 RT gene is as reported by Ratner et al (Ratner et al., Nature,313, 277, (1985)).

It is demonstrated that these specific mutations conferzidovudine-resistance, as an infectious molecular HIV clone containingonly these nucleotide changes is resistant to zidovudine. (See Example4).

From analysis of clinical samples it appears that the sensitivity ofHIV-1 to zidovudine changes over a period of time. It appears thatmutations may occur at any of one or more of the five identified sitesas the time from the onset of treatment with zidovudine advances and itis clear than an HIV-1 sample which carries all five mutations is highlyresistant to zidovudine.

At this time it is not possible to predict any order of occurrence ofthese mutations, although particular attention is being focussed on thetwo nucleotides of the wild-type DNA sequence (or its corresponding RNA)or to the two nucleotides of the mutant DNA sequence (or itscorresponding RNA) set forth in FIG. 1 at the 2772- and 2773- positions.

According to a first aspect of the invention there is provided a methodfor assessing the sensitivity of an HIV-1 sample to zidovudine, whichcomprises:

-   -   (i) isolating nucleic acid from the sample,    -   (ii) hybridising an oligonucleotide to the nucleic acid, the        oligonucleotide being complementary to a region of the wild-type        DNA sequence (or its corresponding RNA) or to a region of the        mutant DNA sequence set forth in FIG. 1 (or its corresponding        RNA) and terminating at the 3′-end with the nucleotide in the        2328-, 2338-, 2772-, 2773- or 2784-position,    -   (iii) attempting polymerization of the nucleic acid from the 3′        end of the oligonucleotide,    -   (iv) ascertaining whether or not an oligonucleotide primer        extended product is present.

It is possible to use genomic DNA or RNA isolated from HIV-1 samples inthis methodology. Suitable cells for supporting the growth of HIV-1,such as MT-2 cells, are firstly infected with an HIV-1 isolate andincubated for a period of time. The cells are recovered bycentrifugation. DNA can then be isolated by digestion of the cells withproteinase K in the presence of EDTA and a detergent such as SDS,followed by extraction with phenol (see Example 1 for the methodologyused by the inventors for the isolation of HIV-1 DNA).

Well-known extraction and purification procedures are available for theisolation of RNA from a sample. RNA can be isolated using the followingmethodology. Suitable cells are again infected and incubated for aperiod of time. The cells are recovered by centrifugation. The cells areresuspended in an RNA extraction buffer followed by digestion using aproteinase digestion buffer and digestion with proteinase K. Proteinsare removed in the presence of a phenol/chloroform mixture. RNA can thenbe recovered following further centriguation steps. (Maniatis, T., etal, Molecular Coning, A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory Press, (1989)).

Although it is possible to use unamplified nucleic acid, due to therelative scarcity of nucleic acid in an HIV-1 sample it is preferably toamplify it. Nucleic acid may be selectively amplified using thetechnique of polmerase chain reaction (PCR), which is an in vitro methodfor producing large amounts of a specific nucleic acid fragment ofdefined length and sequence form small amounts of a template.

The PCR is comprised of standard reactants using Mg²⁺ concentration,oligonucleotide primers and temperature cycling conditions foramplification of the RT gene using the primers. The primers are chosensuch that they will amplify the entire RT gene or a selected sequencewhich incorporates nucleotides corresponding to a region of thewild-type DNA sequence of HIV-1 between the nucleotides at the 2328- and2784-positions set forth in FIG. 1. Example 2 provides a description ofPCR used to amplify target nucleic acid.

RNA cannot be amplified directly by PCR. Its corresponding cDNA must befirst of all synthesised. Synthesis of cDNA is normally carried out byprimed reverse transcription using oligo-dT primers. Advantageously,primers are chosen such that they will amplify the nucleic acid sequencefor RT or a selected sequence which incorporates nucleotidescorresponding to the region of RNA corresponding to the wild-type DNAsequence or to the region of the mutant DNA sequence set forth in FIG. 1between the nucleotides 2328- and 2784-. This could be achieved bypreparing an oligonucleotide primer which is complementary to a regionof the RNA strand which is up-stream of the corresponding sequence ofthe wild-type DNA sequence between nucleotides 2328- and 2784-. cDNAprepared by this methodology (see Maniatis, T., et al., supra.) can thenbe used in the same way as for the DNA already discussed.

The next stage of the methodology is to hybridise to the nucleic acid anoligonucleotide which is complementary to a region of the wild-type DNAsequence (or its corresponding RNA) or to a region of the mutant DNAsequence (or its corresponding RNA) set forth in FIG. 1 and terminatingat the 3′- end with the nucleotide at the 2328-, 2338-, 2772-, 2773- or2784- position.

Conditions and reagents are then provided to permit polymerisation ofthe nucleic acid from the 3′-end of the oligonucleotide primer. Suchpolymerisation reactions are well-known in the art.

If the oligonucleotide primer has at its 3′-end a nucleotide which iscomplementary to a mutant genotype, that is a genotype which has anucleotide change at the 2328-, 2338-, 2772-, 2773- or 2784- positionsas set forth in FIG. 1 then polymerization of the nucleic acid sequencewill only occur if the nucleic acid of the sample is the same as themutant genotype. Polymerisation of a wild type nucleic acid sequencewill not occur or at least not to a significant extent because of amis-match of nucleotides at the 3′- end of the oligonucleotide primerand the nucleic acid sequence of the sample.

If the oligonucleotide primer has at its 3′-end a nucleotide which iscomplementary to the wild-type genotype, that is a genotype which hasthe wild-type nucleotide at the corresponding 2328-, 2338-, 2772-, 2773-or 2784- position as set forth in FIG. 1, then there will bepolymerisation of a nucleic acid sequence which is wild-type at thatposition. There will be no polymerisation of a nucleic acid which has amutant nucleotide at the 3′-position.

The preferred length of each oligonucleotide is 15-20 nucleotides. Theoligonucleotide can be prepared according to methodology well known tothe man skilled in the art (Koster, H., Drug Research, 30 p548 (1980);Koster, H., Tetrahedron Letters p1527 (1972); Caruthers, TetrahedronLetters, p719, (1980); Tetrahedron Letters, p1859, (1981); TetrahedronLetters 24, p245, (1983); Gate, M., Nucleic Acid Research, 8 p1081,(1980)) and is generally prepared using an automated DNA synthesisersuch as an Applied Biosystems 381A synthesiser.

It is convenient to determine the presence of an oligonucleotide primerextended product. The means for carrying out the detection is by usingan appropriate label.

The label may be conveniently attached to the oligonucleotide primer orto some other molecule which will bind the primer extendedpolymerisation product.

The label may be for example an enzyme, radioisotope or fluorochrome. Apreferred label may be biotin which could be subsequently detected usingstreptavidin conjugated to an enzyme such as peroxidase or alkalinephosphatase. The presence of an oligonucleotide primer extendedpolymerisation product can be detected for example by running thepolymerisation reaction on an agarose gel and observing a specific DNAfragment labelled with ethidium bromide, or Southern blotted andautoradiographed to detect the presence or absence of bandscorresponding to polymerised product. If a predominant band is presentwhich corresponds only to the labelled oligonucleotide then thisindicates that polymerisation has not occurred. If bands are present ofthe correct predicted size, this would indicate that polymerisation hasoccurred.

For example, DNA isolated from patients' lymphocytes as described hereinis used as a template for PCR amplification using syntheticoligonucleotide primers which either match or mis-match with theamplified sequences. The feasibility of PCR in detecting such mutationshas already been demonstrated. PCR using the Amplification RefractoryMutation system (“ARMS”) (Newton, C. R., et a. Nucleic Acids Research,17, p.2503, (1989)) Synthetic oligonucleotides are produced that annealto the regions adjacent to and including the specific mutations suchthat the 3′ end of the oligonucleotide either matches or mismatches witha mutant or wild-type sequence. PCR is carried out which results in theidentification of a DNA fragment (using gel electrophoresis) where amatch has occurred or no fragment where a mismatch occurred.

For example, using the 2 oligonucleotides below as PCR primers:

-   5′-ATG TTT TTT GTC TGG TGT GGT-3′- (1) OR-   5′-ATG TTT TTT GTC TGG TGT GAA-3′- (2)    plus the common oligonucleotide primer:-   “B”-5′- GGA TGG AAA GGA TCA CC-3′    it is possible to distinguish between sensitive and resistant virus.    DNA is extracted from HIV-1 infected T-cells as described herein and    subjected to “ARMS” PCR analysis using these primers. If the virus    is sensitive a 210 bp fragment is generated with oligonucleotide    B+(1) but not with B+(2). By contrast, if the virus is zidovudine    resistant a 210 bp fragment is generated with B+(2) but not with    B+(1).

The presence of a fragment is identified by using an oligonucleotideprimer as described above, i.e. by attempting polymerisation using anoligonucleotide primer which may be labelled for the amplified DNAfragment under stringent conditions which only allow hybridisation ofcomplementary DNA (the only difference is that differentialhybridisation does not have to be performed as fragments of DNAamplified by the “ARMS” method will be the same whether derived frommutant or wild-type DNA, so a common oligonucleotide can be used todetect the presence of these fragments. The sequence of such anoligonucleotide is derived from a DNA sequence within the DNA fragmentthat is conserved amongst HIV-1 strains).

The above PCR assay may be adapted to enable direct detection ofmutations associated with zidovudine resistance in DNA from PBL samplesfrom infected individuals that have not been cultured to obtain virus.As this material generally contains considerably less HIV-1 DNA thanthat in infected lymphoid cultures a “double PCR” (or nested set)protocol can be used (Simmonds, P., Balfe, P. Peutherer, J. F., Ludlam,C. A., Bishop, J. O. and Leigh Brown, A. J., J. Virol., 64, 864-872,(1990)) to boost the amount of target HIV-1 RT DNA signal in thesamples. The double PCR overcomes the problem of limited amplificationof a rare template sequence. Initially a fragment may be amplified fromwithin the RT region which encompasses all the commonly observedmutations. A small amount of the pre-amplified material may be used inthe second PCR with primer pairs designed to allow discrimination ofwild type and mutant residues.

A suitable test kit for use in an assay to determine the resistancestatus of an HIV-1 sample to zidovudine which makes use of a methodologyaccording to the first aspect of the invention, comprises anoligonucleotide being complementary to a region of the wild-type DNAsequence (or its corresponding RNA) or to a region of the mutant DNAsequence set forth in FIG. 1 (or its corresponding RNA) and terminatingat the 3′-end with the nucleotide in the 2328-, 2338-, 2772-, 2773- or2784- position, other materials required for polymerisation of thenucleic acid from the 3′-end of the oligonucleotide and means fordetermining the presence of an oligonucleotide primer extended product.Such other materials include appropriate enzymes, buffer and washingsolutions, and a label and a substrate for the label if necessary. IfPCR is used to amplify nucleic acid then additional materials such asappropriate oligonucleotide primers which will amplify a region of thewild-type DNA sequence (or its corresponding RNA) or a region of themutant DNA sequence set forth in FIG. 1 (or its corresponding RNA)containing one or more of the nucleotides at the 2328-, 2338-, 2772-,2772- or 2784- position, appropriate enzymes and dNTP's should beincluded.

In a second aspect of the invention there is provided a method fordetermining the sensitivity of an HIV-1 sample to zidovudine whichcomprises:

-   -   (i) isolating the nucleic acid from the sample,    -   (ii) hybridising the nucleic acid with an oligonucleotide being        complementary to a region of the wild-type DNA sequence (or its        corresponding RNA) or to a region of the mutant DNA sequence set        forth in FIG. 1 (or its corresponding RNA) containing one or        more of the nucleotides at the corresponding 2328-, 2338-,        2772-, 2773- and/or 2784- position,    -   (iii) ascertaining whether or not any of the resulting hybrids        of the oligonucleotide and nucleic acid have complementary        nucleotides at one of these positions.

Preferably the oligonucleotide is so designed to form a perfectlymatched hybrid with its complement.

Nucleic acid (DNA or RNA) is isolated from a sample by theaforementioned methods as described for the first aspect of theinvention.

Similarly, PCR may be used to amplify the RT DNA (or its correspondingRNA) or preferably to amplify a region of the RT DNA (or itscorresponding RNA) which incorporates DNA (or its corresponding RNA)containing one or more of the nucleotides at the 2328-, 2338-, 2772-,2775- and/or 2784- position (see Example 2).

In the second stage of this methodology the nucleic acid is then used tohybridise to oligonucleotides complementary to a region of the wild-typeDNA sequence (or its corresponding (RNA) or to a region of the mutantDNA sequence set forth in FIG. 1 (or its corresponding RNA) containingone or more of the nucleotides at the aforementioned positions.

The oligonucleotide may be of any length depending on the number ofnucleotide positions of interest which are being examined. If theoligonucleotide is designed to include a nucleotide at only one positionof interest then this nucleotide is preferably at or close to the centreposition of the oligonucleotide.

For example, referring to FIG. 1, one oligonucleotide probe fordetection of the mutation at nucleotide 2328- would be complementary tothe mutated RT gene sequence and would include at its nucleotidecorresponding to nucleotide 2328- the nucleotide complementary to themutated 2328- nucleotide. A second oligonucleotide probe for the wildtype HIV-1 RT would include at its nucleotide corresponding tonucleotide 2328- the nucleotide complementary to the wild-type 2328-nucleotide. Oligonucleotide probes designed to detect one or more of themutations referred to in FIG. 1 are used to detect a specific mutationor that there has been a mutation.

In order to ascertain whether or not the oligonucleotide and nucleicacid sequence have formed a matched hybrid, specific hybridisationconditions are set so that a hybrid is only formed when the nucleotideor nucleotides at one or more of the 2328-, 2338-, 2772-, 2773- and2784- position is or are complementary to the corresponding nucleotideor nucleotides of the oligonucleotide which either permits hybridisationor no hybridisation. It is important to establish for example thetemperature of the reaction and the concentration of salt solutionbefore carrying out the hybridisation step to find conditions that arestringent enough to guarantee specificity (Maniatis, T., et al.,Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbourlaboratory Press, (1989)). If the oligonucleotide probe has a DNAsequence which is complementary to a wild-type nucleic acid sequence atone or more of its nucleotides corresponding to the 2328-, 2338-, 2772-,2773- or 2784- position of the DNA sequence of FIG. 1 then thisoligonucleotide will hybridise perfectly to wild-type nucleic acid. Ifthere is no hybridisation then this would suggest that the nucleic acidisolated from the sample contains one or more mutations.

If the oligonucleotide probe has a DNA sequence which is complementaryto a mutant nucleic acid sequence at one or more of its nucleotidescorresponding to the 2328-, 2338-, 2772-, 2773- or 2784- positions ofthe DNA sequence of FIG. 1 then this oligonucleotide will hybridiseperfectly to mutant nucleic acid. If there is no hybridisation thiswould suggest that the nucleic acid isolated from the sample contains nosuch mutation or mutations. The oligonucleotide probes may be labelledas a means of detection as for the first aspect of the invention.

The hybridisation and subsequent removal of non-hybridised nucleic acidsare performed under stringent conditions which only allow hybridisationof the complementary DNA and not the oligonucleotide containing amismatch (i.e. oligonucleotide specific hybridisation as described forthe detection of sickle cell mutation using the β-globin or HLA-DQα gene(Saikt, R. K., et al., Nature, 324, p163, (1986)), the activated Rasgene (Ver Laan-de, Vries, M., et al., Gene, 50, 313, (1986)) andβ-thalassaemia Wong, C., et al., Nature, 330, p384, (1987)).

The hybridisation may be carried out by immobilisation of the RT nucleicacid sequence onto nitrocellulose, nylon or other solid matrix (eg.dot-blot). It is convenient to determine the presence of an hybrid byusing the means of a label. For example, the chemically synthesisedoligonucleotide probes can be suitably labelled using enzyme,radiosotope or fluorochrome. A preferred label may be biotin which couldbe subsequently detected using streptavidin conjugated to an enzyme suchas peroxidase or alkaline phosphatase.

Alternatively the hybridisation may be carried out by immobilisation ofthe chemically synthesised oligonucleotides referred to above, which areunlabelled, onto a solid support referred to above and subsequentlyhybridisation by a labelled RT nucleic acid sequence as describedpreviously.

In both situations described above for hybridisation suitable controlreactions will be incorporated to determine that efficient hybridisationhas occurred. (eg the hybridisation of oligonucleotides to acomplementary oligonucleotide).

Results would be readily interpreted as the isolated nucleic acid wouldhybridise to either the wild type oligonucleotide or the mutantoligonucleotide.

A suitable test kit for use in an assay to determine the sensitivity ofan HIV-1 sample to zidovudine which makes use of a methodology accordingto the second aspect of the invention comprises an oligonucleotide beingcomplementary to a region of the wild-type DNA sequence (or itscorresponding RNA) or to a region of the mutant DNA sequence set forthin FIG. 1 (or its corresponding RNA) containing one or more of thenucleotides at the corresponding 2328-, 2338-, 2772-, 2773- and/or 2784-position, other materials required to permit hybridisation. Suchmaterials include appropriate buffers and washing solutions and a labeland a substrate for the label if necessary. Normally the oligonucleotidewould be labelled. If PCR is used to amplify nucleic acid prior tohybridisation then additional materials such as appropriateoligonucleotide primers which will amplify a region of the wild-type DNAsequence (or its corresponding RNA) or a region of the mutant DNAsequence set forth in FIG. 1 (or its corresponding RNA) containing oneor more of the nucleotides at the 2328-, 2338-, 2772-, 2773- or 2784-position, appropriate enzymes and dNTP's (deoxy nucleotidetriphosphates) should be included.

In one alternate format of the assay, the dNTP's in the amplificationmay or may not be coupled to a detector molecule such as a radiosotope,biotin, fluorochrome or enzyme.

It is also possible to detect zidovudine resistant mutations in theHIV-1 RT RNA isolated from clinical samples using an RNA amplificationsystem. Using the methodology described by Guatelli et al. (Proc. Natl.Acad. Sci, (USA), 8/7, 1874-1878, (March 1990)) a target nucleic acidsequence can be replicated (amplified) exponentially in vitro underisothermal conditions by using three enzymatic activities essential toretroviral replication: reverse transcriptase, RNase H and aDNA-dependent RNA polymerase. Such a methodology may be employedfollowed by an hybridisation step to distinguish mutant from wild-typenucleotides as discussed previously.

The following examples are for illustration only and are not intended tolimit the invention in any way. In the accompanying drawings:

FIG. 1 shows the nucleotide changes at the five positions in HIV-1 RTthat confer resistance to zidovudine. Numbering of the nucleotides is asreported by Rather et al., Nature, 313, 277, (1985).

FIG. 2 shows the amino acid changes at the four positions in HIV-1 RTthat confer resistance to zidovudine.

FIG. 3 shows the scheme used to amplify and clone HIV RT.

FIG. 4 shows mutational analysis of HIV RT using the polymerase reactionin which a. shows a schematic representation of the HIV-1 genone showingthe location and orientation of oligonucleotide primers in the RT codingregion used in PCR reactions. Primers 2W and 2M anneal to the sequenceat codon 70 (indicated as

) and are used separately with primer A to generate a DNA fragment of227 bp. Primers 3W and 3M anneal to sequence at codon 215 (indicated as

) and are used separately with primer B to generate a 210 bp fragment);b. shows details of PCR primers 2W and 2 M initiating DNA synthesis atcodon 70. The sequence of each primer is shown with variation at the 3′end highlighted in bold letters where a correct match forms with targetHIV DNA. In this situation Taq DNA polymerase can initiate DNA synthesisfrom the 3′ end of the primer

). Nucleotide sequence numbers according to Ratner at el., supra areshown in parenthesis. c. shows PCR primers 3W and 3M initiating DNAsynthesis at codon 215. Sequence variation at the 3′ end is highlightedin bold letters where a correct match is formed with target HIV DNA.

FIG. 5 shows a direct analysis of RT codon 215 in DNA from non-culturedPBL samples. Early and late PBL samples obtained from three patientswith AIDS (ptA, ptB and ptL) receiving zidovudine therapy were subjectedto “double” PCR to discriminate between wild type and mutant residues.Oligonucleotide primers A and NE1 were used in the initial PCR,producing a 768 bp fragment (see FIG. 4) and material from thisamplification was used in the second PCR where primer B was pairedseparately With primer 3W (WT) or 3M (M) to discriminate between wildtype and mutant nucleotides at condon 215. Produces (15 μl) from thesecond PCR were separated on a 1.5% agarose gel and stained withethidium bromide. The numbers above pairs of lanes on the gel refer tothe treatment time which each sample was obtained (in months). Allpatients received 1200 mg of zidovudine per day throughout the treatmentperiod.

EXAMPLE 1 Isolation of HIV1 DNA

The isolation of HIV-1 was achieved according to methodology reported byLarder et al. PBL samples were prepared by separation on“Ficoll-hypaque” gradients and co-cultivated directly with MT-2 cells(approximately 10⁶ of each) after pre-stimulation for 24 to 72 hourswith phytohaemag-glutinin (3 μg/ml). In many cases PBLs were culturedafter long-term frozen storage. Cultures were maintained in RPMI 1640supplemented with 10% fetal bovine serum, antibiotics, 2% interleukin-2,and polybrene (2 μg/ml) and expanded by addition of fresh MT-2 cellswhen a cytopathic effect was observed (between 4 and 14 days). HIVreplication was confirmed by the detection of p24 antigen in culturesupernatants (p24 antigen detection kit, Abbott, Chicago, Ill.). Viruswere prepared from infected cultures and stored in aliquots at −70° C.All drug sensitivities were determined with virus pools that had beenpassaged no more than twice from original cultures. (Larder, B. A.,Darby, G., Richman, D., Science, 243, 1731, (1989)).

DNA was extracted from MT-2 cells infected with HIV isolates as follows:2×10⁶ MT-2 cells were infected with HIV-1 at a multiplicity of approx.0.1 TCID₅₀/cell and incubated at 37° C. for 3-4 days in RPM1 - 1640medium supplemented with 10% foetal calf serum, polybrene (2 μg/ml) andantibiotics (sRPMI/10). The reference to Science, 246, 1155-1158 (1989)is made to permit the reader to obtain more information about MultipleMutations in HIV-1 Reverse Transcriptase. The methodology for theextraction of DNA as provided in this Example is complete (Larder, B.A., Kemp, S. D., Science, 246, 1155-1158, (1989)).

DNA was extracted from the cell pellet by lysis in 0.4% SDS, 25 mMTris-HCl, pHS, 25 mM, EDTA, and 150 mM NaCl and followed by Proteinase K(1 mg/ml) digestion for 1 hr at 50° C. Nucleic acid was recovered byphenol/chloroform extraction and ethanol precipitation, was treated withRNase A (10 μg/ml) and DNA was subjected to further phenol/chloroformextraction before ethanol precipitation.

EXAMPLE 2 Amplification and Cloning of HIV Reverse Transcriptase

The complete RT coding region (1.7 kb) was obtained for each HIV-1isolate using polymerase chain reaction (PCR) amplification of infectedcell DNA.

PCR is a powerful technique for selectively amplifying DNA or RNAsequences. It is an in vitro method for producing large amounts of aspecific DNA fragment of defined length and sequence from small amountsof a template. PCR is based on the enzymatic amplification of a DNAfragment that is flanked by two oligonucleotide primers that hybridiseto opposite strands of the target sequence. The primers are orientedwith their 3′ ends pointing towards each other. Repeated cycles of heatdenaturation of the template, annealing of the primers to theircomplementary sequences and extension of the annealed primers with a DNApolymerase result in the amplification of the segment defined by the 5′ends of the PCR primers. Since the extension product of each primer canserve as a template for the other primer, each cycle essentially doublesthe amount of the DNA fragment produced in the previous cycle. Thisresults in the exponential accumulation of the specific target fragment,up to several millionfold in a few hours. The method can be used with acomplex template such as genomic DNA and can amplify a single-copy genecontained therein. It is also capable of amplifying a single molecule oftarget in a complex mixture of RNAs or DNAs and can, under someconditions, produce fragments up to ten kilobase pairs long. (White, T.J., Arnheim, N., and E. Hich, A., Technical Focus Vol. 5, No. 6, p185June 1989).

A diagramatic representation of the scheme used to amplify and clone HIVRT is shown in FIG. 3. Total DNA was extracted from cells infected withHIV isolates and the entire 1.7 kb fragment of the RT coding region wasobtained by PCR (Saiki, R. K., et al. Science 239, 487 (1988)) HIV-1 DNAwas obtained as in Example 1. Approximately 1 μg of this DNA was usedper PCR reaction to amplify the complete RT coding region. Each reactionmix (100 μl) contained 25 mM KCl, 1.5 mM MgCl₂, 50 mM tris-HCl, pH8.3,0.1 mg/ml bovine serum albumin, 0.2 mM each of dATP, dGTP, dCTP, TTP,0.25 μg each primer and 2.5 units of Taq DNA Polymerase (Perkin-ElmerCetus). These mixtures were heated at 100° C. for 2 mins prior toaddition of Taq DNA polymerase, overlaid with 100 μl light mineral oiland subjected to 30 cycles consisting of a denaturation step (1 min 30secs, 94° C.) primer annealing (2 min, 37° C.) and DNA synthesis (10min, 72° C.) using a Perkin-Elmer Cetus DNA thermal cycler. Theoligonucleotide primers, made using an Applied Biosystems 381Asynthesiser, were as follows: at the 5′ end of RT,5′-TTGGACTTTGAATTCTCCCATTAG-3′ and 5′-TGTACTTTGAATTCCCCCATTAG-3′ (twoprimers were used to accommodate sequence variation seen in this region)and at the 3′ end, 5′-CTTATCTATTCCATCTAGAAATAGT-3′.

HIV RT obtained by PCR amplification was digested with EcoRI and XbaI,whose recognition sites were built into the 5′ and 3″ ends by the PCRprimers, fragments were purified from agarose gels and ligated with thereplicative form (RF) of M13mp18, previously digested with EcoRI andXbaI. HIV RT (1.7Kb) obtained by PCR amplification (Saiki, R. K., et al.Science 239, 487 (1988)) was digested with EcoRI and XbaI (BRL),purified from agarose gels and ligated with the M13 vector mp18pre-digested with EcoRI and XbaI (see FIG. 3). The ligation mixture wasused to transform E. coli (strain TG-1) made competent as described (D.Hanahan, J. Mol. Biol. 166, 557 (1983)). Single-stranded DNA wasprepared from these constructs for nucleotide sequencing. Clones able toexpress functional RT enzyme were sequenced using the well-knowndideoxynucleotide chain-termination method (Sanger, F., Nicklen, S.,Coulson, A. R., Proc. Natl. Acad. Sci., USA, 74, 5463, (1977)). Table 1shows details of the RT clones obtained by this procedure and lists theHIV isolates from which they were derived. The complete sequence of 6 RTclones was obtained (sensitive and resistant isolate pairs the threeindividuals) and partial sequence data were obtained from additionalisolates.

PCR primers can be designed to incorporate a restriction enzymerecognition site such as EcoRI at the 5′ end of the HIV-1 RT gene and arestriction enzyme such as Xbal at the 3′ end. Digestion of eachfragment with restriction enzymes allows subsequent cloning into asuitable vector such as an M13 mp18 based vector.

EXAMPLE 3 Properties of HIV-1 Isolates and M13 RT Clones Derived fromThem. Mutations in HIV-1 RT conferring zidovudine Resistance

HIV isolates derived from untreated and treated individuals are shown inTable 1 with duration of therapy at the time they were obtained and thezidovudine sensitivity. Where multiple isolates were obtained from thesame individual, isolates are lettered in temporal order. Fifty per centinhibitory dose (ID₅₀) values were obtained by plaque-reduction assaywith HT4-6C cell monolayers Inhibition of plaque formation (foci ofmultinucleated giant cells) was determined by infecting monolayers ofHeLa HT4-6C cells with cell-free HIV preparations. The input inoculumwas adjusted to give 100 to 300 plaques per well (in 24-well plates) inthe no-drug control cultures. Virus was allowed to absorb for one hourat 37° C. prior to the addition of inhibitor in the culture medium(Dulbecco's modification of Eagle's medium, containing 5% fetal bovineserum plus antibiotics). After 3 days of incubation, monolayers werefixed with 10% formaldehyde and stained with 0.25% crystal violet tovisualize plaques. This staining procedure revealed obvious individualdense foci of multinucleated giant cells. ID₅₀ values were deriveddirectly from plots of 50% plaque reduction cersus inhibitorconcentration. (Larder, B. A. Darby. G., Richman, D. D., Science, 243,1731, (1989); Chesebro, B. D., Wehrly, K., J. Virol., 62, 3779 (1988).).

The amino acid residue at four positions in HIV-1 RT important forzidovudine resistance are illustrated for HIV isolates. Wild-typeresidues at the positions of interest are as follows: Asp 67, Lys 70,Thr 215 and Lys 219.

Comparison of predicted complete amino acid RT sequences from sensitiveand highly-resistant isolate pairs obtained from the same individuals(patients A012, AO18 and P022) revealed substantial differences ofbetween 14-17 residues. In each case however, we identified amino acidchanges at four residues (Asp 67→Asn, Lys 70→Arg, Thr 215→Phe or Tyr,and Lys 210→Gln) in the highly resistant isolates not seen in thesensitive counterparts (FIG. 2). Analysis of sensitive andhighly-resistant isolate pairs from a further two individuals (patientsA036 and PO26) revealed the same mutations at positions 67, 70 and 215,whilst retaining the wild-type Lys at residue 219 (FIG. 2). In caseswhere multiple M13 clones were sequenced from an isolate only minorsequence variation was seen. It was interesting that published HIV-1 RTsequences showed absolute conservation of all four residues in everystrain (G. Myers et al., (eds) Human retroviruses and AIDS: acompilation and analysis of nucleic acid and amino acid sequences(Theoretical Biology and Biophysics, Los Alomos, N.M.) II-22 (1989),strongly implicating these mutations in zidovudine-resistance.

TABLE 1 DURATION OF ZIDOVUDINE RT ACTIVITY THERAPY SENSITIVITY M13 CLONEAMINO ACID RESIDUES IN RT HIV ISOLATE (Months) (ID₅₀ μM) (% Control) 6770 215 219 A012B 1 0.01 20 Asp Lys Thr Lys A012D 26 2 85 Asn Arg Phe GlnA018A 0 0.01 62 Asp Lys Thr Lys A018C 14 2.3 79 Asn Arg Tyr Gln A036B 20.01 47 Asp Lys Thr Lys A036C 11 0.6 52 Asp Lys Tyr Lys A036D 20 5.6 46Asn Arg Tyr Lys P022A 1 0.01 20 Asp Lys Thr Lys P022C 16 1.4 67 Asn ArgPhe Gln P026A 0 0.01 76 Asp Lys Thr Lys P026B 11 2.8 78 Asn Arg Tyr LysP035A 6 0.56 55 Asp Lys Tyr Lys

EXAMPLE 6 Zidovudine-sensitivity of HIV Valiant Created by Site-DirectedMutagensis

To text whether multiple mutations in the RT gene could account for highlevel resistance, an infectious molecular clone of HIV containing onlythe four mutations described above was constructed (the isolate AO12D)to assess the sensitivity of virus produced from this clone bytransfection of T-cells. A 2.55 kb fragment of the HIV Pol gene frominfectious clone HXB2-D (Fisher, A. G., et al., Nature 316, 262, 1985)inserted into the M13 vector mp19, was used as a target forsite-directed mutagenesis (Larder, B. A., Kemp, S. D., Purifoy, D. J.M., Proc. Natl. Acad. Sci., USA, vol 86; p.4803 (1989)

Specific nucleotide changes were simultaneously introduced into the RTgent using two synthetic oligonucleotides and mutations were confirmedby nucleotide sequencing (Sanger, F., Nicklen, S. A., Coulson, A. R.,Proc. Natl. Acad. Sci., USA. 74, 5463, (1977)). To reconstruct thefull-length clone, a 1.9 kb BalI restriction fragment containing themutations in RT was removed from the pol gene M13 clone and transferredinto HXB2-D (Larder, B. A., Kemp, S. D., Purifoy, D. J. M., Proc. Natl.Acad. Sci, USA, Vol. 86, p4803, (1989)). DNA prepared from wild-type andmutant infectious clones was then used to transfect the T-celllymphoblastoid line, MT-4 (Harada, S., Koyanagi, U., Yamomoto, N.,Science, 229, 563, (1985)), by electroporation. Virus-induced CPE wasobserved in each culture at similar times (after 2-4 days) and virusstocks were prepared 6-7 days post transfection. Wild type and mutantHIV isolates (HXB2-D and HIVRTMC respectively) were titrated by plaqueassay in the HeLa-CD4 ⁺ cell line, HT4-6C, and then tested forsensitivity to zidovudine by plaque-reduction assay in HT4-6C cells(Larder, B. A., Darby, G., Richman, D. D., Science, 243, 1731, (1989);Chesebro. B. D., Wehrly, K., J. Virol., 62 3779, (1988)). The results ofthese experiments as shown in Table 2 clearly demonstrate that themutant virus constructed by site-directed mutagenesis was highlyresistant to zidovudine. The ID₅₀ value for HIVRTMC increased about100-fold compared to wild-type virus and the magnitude of resistance wassimilar to that of naturally occurring HIV isolates containing similarmutations in the RT gene (Table 2).

TABLE 2 ZIDOVUDINE SENSITIVITY FOLD HIV ISOLATE (Mean ID₅₀, μM) INCREASEHXB2.D 0.013 (0.005) 1 HIVRTMC 1.28 (0.24) 98 A012B 0.013 (0.005) 1A012D 2.56 (1.03) 197

EXAMPLE 5 Selective amplification of HIV DNA to detect resistancemutations

DNA for PCR was obtained from MT-2 cells (2×10⁶) infected with HIVisolates at a multiplicity of 0.1 TCID₅₀/ml (fifty per cent tissueculture infectious dose per ml, as determined by terminal dilution ofvirus stocks in MT-2 cells) and incubated at 37° C. for 3-4 days in RPMI1640 medium supplemented with 10% fetal bovine serum, polybrene (2μg/ml) and antibiotics. DNA was extracted from cell pellets by lysis in0.4% SDS, 25 mM Tris HCl, pHS, 25 mM EDTA and 150 mM NaCl. Afterdigestion with proteinase K (1 mg/ml, 1 h at 50° C.) DNA was recoveredby phenol extraction and ethanol precipitation. Approximately 1 μg ofthis material was used per PCR reaction (100 μl) which contained: 25 mMKCl, 50 mM Tris HCl, pH8.3, 0.1 mg/ml bovine serum albumin (BSA), 0.2 mMeach of dATP, dGTP, dCTP and dTTP, 0.25 μg of each oligonucleotideprimer with the following concentrations of MgCl₂ for specificcombinations of primer pairs:

-   -   Primer A with 1W and 1M (to analyse codon 67): 2 mM MgCl₂    -   Primer A with 2W and 2M (to analyse codon 70): 5 mM MgCl₂    -   Primer B with 3W and 3M (to analyse codon 215): 1.8 mM MgCl₂    -   Primer B with 4W and 4M (to analyse codon 219): 1.5 mM MgCl₂

Reaction mixtures were heated at 100° C. for 2 mins prior to addition ofTaq DNA polymerase (2.5 units, Perkin-Elmer Cetus), overlaid with 100 μllight mineral oil and subjected to 30 cycles consisting of adenaturation step (1 min, 94° C.), primer annealing (30 sec, 40° C. forreactions to analyse codon 70 (primer A with 2W and 2M) and codon 219(primer B with 4W and 4M) or 45° C. for reactions to analyse codon 67(primer A with 1W and 1M) and codon 215 (primer B with 3W and 3M) andDNA synthesis (30 sec, 72° C.) using a Perkin-Elmer Cetus DNA thermalcycler. The oligonucleotide primers (FIG. 4) were synthesised using anApplied Biosystems 381A machine. 10 μl of each reaction mixture was runon 1.5% TBE agarose gels and DNA was visualized by ethidium bromidestaining. The sequence of PCR primers 2W, 2M, 3W and 3M are shown inFIG. 4b and c. The sequences of the other oligonucleotide primers wereas follows:

primer A, 5′-TTCCCATTAGTCCTATT-3′; primer B, 5′-GGATGGAAAGGATCACC-3′;primer IW, 5′-TTTTCTCCATTTAGTACTGAC-3′; primer IM,5′-TTTTCTCCATCTAGTACTGAT-3′; primer 4W, 5′-AGGTTCTTTCTGATGTTTTAT-3′;primer 4M, 5′-AGGTTCTTTCTGATGTTTTAG-3′.

EXAMPLE 6 Construction of HIV variants with defined Combinations ofmutations in RT

Variants were constructed to mimic mutants identified by DNA sequenceanalysis of clinical isolates and in addition, a number of mutants weremade containing combinations of changes in RT not yet seen. Thenucleotide sequence of RT derived from the proviral clone HXB2-D(Fisher, A. G., Collatti, E., Ratner, L. Gallo, R. C. and Wong Staal,F., Nature (London), 316, 262-265, (1985)) was initially altered by sitedirected mutagenesis. A pol gene DNA fragment from M13 clones containingaltered RT was mixed with HXB2-D that had a Bali restriction enzymefragment encompassing the RT region removed. These mixtures were used toco-transfect MT-2 cells by electroporation in order to allow formationof infectious virus variants through homologous recombination. Mutationsin RT were created by site-directed mutagenesis of the previouslydescribed M13 clone mpRT1/H, which contains a 2.55 kb Bgl II to EcoRIfragment of the wild type HIV pol gene (Larder, B. A., Kemp, S. D., andPurifoy, D. J. M., Proc. Natl. Acad. Sci., (USA), 86, 4803-4807 (1989)).All mutants were verified by nucleotide sequence analysis (Sanger, F.,Nicklen, S, and Coulson, A. R. Proc. Natl. Acad. Sci., USA, 74,5463-5467 (1977)). Wild type virus HXB2 and mutants were derived byhomologous recombination in MT-2 cells (Clavel, F., Hogan, M. D.,Willey, R. L., Strebel, K., Martin, M. A. and Repaske, R., J. Virol.,63, 1455-1459 (1989); Srinivasan, A., et al., Proc. Natl. Acad. Sci.,(USA) 86, 6388-6392 (1989)) by co-transfecting a wild type full lengthHIV clone missing most of RT and the pol gene fragment from mpRT1/H M13clones. Wild type infectious clone HXB2-D (Fisher, A. G., Collati, E.,Rather, L., Gallo, R. C., and Wong-Staal, F., Nature (London), 316,262-265, (1985)) was digested with Msc I (Bal I), releasing a 1.9 kbfragment comprising the majority of RT (Ratnet, L. et al., Nature(London) 313, 277-284, (1985)) and the large fragment was purified fromagarose gels. The replicative form (double stranded DNA) of mpRT1/H ormutated clones were digested with EcoRI and Hind III to linearize thepol gene fragment and 5 μg of each was mixed with 5 μg of gel purifiedHXB2-D DNA (with the Msc I fragment removed). Each mixture was used totransfect MT-2 cells by electroporation as described (Larder, B. A.,Kemp, S. D. supra.) and infectious virus (recovered in culturesupernatants at around 14 days post transfection) was stored in aliquotsat −70° C. The genotype of mutant viruses were verified by DNA sequenceanalysis of RT cloned from these isolates by PCR. Virus isolatesrecovered from these cultures were tested for sensitivity to zidovudineby plaque reduction assay in HT4-6C cells. Virus variants were generallymore resistant with increasing numbers of mutations (table 4), which wasin broad agreement with DNA sequence data obtained from clinicalisolates (table 3). For example, recombinants RTMF (Thr215→Tyr) andRTMC/WT (Asp6-7→Asn, Lys70→Arg) were less resistant to zidovudine thanvariant RTMC/F (Asp67→Asn, Lys70→Arg, Thr215→Tyr) and the degree ofresistance of RTMC/F was about equal to the sum of ID₅₀ values seen withRTMF and RTMC/WT.

TABLE 3 Zidovudine PCR Analysis HIV Months of Sensitivity Codon 70 Codon215 Isolate Therapy (ID₅₀ μM) RT sequence WT M WT M A001A 0 0.03 . . .. + − + − . R₇₀ . . A001B 12 0.11 − + + − N₆₇ R₇₀ . Q₂₁₉ A012B 2 0.01 .. . . + − + − A012D 26 2 N₆₇ R₇₀ F₂₁₅ Q₂₁₉ − + − + A018A 0 0.01 . . .. + − + − A018C 14 4 N₆₇ R₇₀ Y₂₁₅ Q₂₁₉ − + − + A036B 2 0.01 . . . . +− + − A036C 11 0.67 . . Y₂₁₅ . + + − + A036D 20 5.6 N₆₇ R₇₀ Y₂₁₅ . − +− + A025A 24 0.11 . R₇₀ . . − + + − P035A 6 0.18 . . Y₂₁₅ . + − − +P036A 6 0.35 N₆₇ R₇₀ Y₂₁₅ . − + − + Properties of HIV isolates obtainedbefore and during zidovudine therapy Table 4 Zidovudine sensitivity HIVVariant RT Sequence (ID₅₀, μM) Fold Increase HXB2 . . . . 0.01 1 HIVRTMIN₆₇ . . . 0.01 1 HIVRTMJ . R₇₀ . . 0.08 8 HIVRTMF . . Y₂₁₅ . 0.16 16HIVRTMK . . S₂₁₅ . 0.01 1 HIVRTMA2 . . . Q₂₁₉ 0.01 1 HIVRTMC/ N₆₇ R₇₀ .. 0.17 17 WT HIVRTMA3 . . F₂₁₅ Q₂₁₉ 0.22 22 HIVRTMC/ N₆₇ R₇₀ . Q₂₁₉ 0.2828 A2 HIVRTMC/ N₆₇ R₇₀ Y₂₁₅ . 0.35 35 F HIVRTMC N₆₇ R₇₀ F₂₁₅ Q₂₁₉ 1.66166 Zidovudine sensitivity of HIV variants with defined mutations inreverse transcriptase

EXAMPLE 7 Detection of mutations in DNA from non-cultured lymphold cells

The above PCR assay (Example 5) was adapted to enable direct detectionof mutations associated with zidovudine resistance in DNA from PBLsamples from infected individuals that had not been cultured to obtainvirus. As this material generally contains considerably less HIV DNAthan that in infected lymphoid cell cultures, we used a “double PCR” (ornested set) protocol (Simmonds, P., et al., Supra.) to boost the amountof target HIV RT DNA signal in the samples. Therefore, we initiallyamplified a 768b fragment from within the RT region which encompassedall the commonly observed mutations (PCR primers A and NE1 were used inthis amplification, see FIG. 4a). A small amount of this pre-amplifiedmaterial was then used in the second PCR with primer pairs designed toallow discrimination of wild type and mutant residues. Results of atypical analysis of amino acid residue 215 are shown in FIG. 5. PBLsamples were obtained from three individuals with AIDS prior toinitiation of zidovudine therapy and after 12-17 months of therapy. Thepre-treatment samples all appeared to be wild type, whilst thepost-treatment initiation samples were either mutant or appeared amixture of both (FIG. 5).

Detection by PCR of mutations in DNA from non-cultured PBL samples

DNA was extracted from about 2×10⁶ PBLs as described above for MT-2 cellcultures. 0.5-1 μg of this material was used in the initial PCR (100 μl)which contained 25 mM KCl, 1.8 mM MgCl₂, 50 mM Tris HCl, pH 8.3, 0.1mg/ml BSA, 0.2 mM each of dATP, dGTP, dCTP and dTTP plus 0.25 μg of eachprimer (primer A and primer NE1). Reaction mixtures were processed asdescribed in Example 5, and subjected to 30 cycles of 1 min 94° C., 1min 45° C. and 2 min 72° C. Following amplification, samples wereextracted by the addition of 100 μl chloroform and 0.5 μl of each wasadded directly to reaction tubes containing all constituents of therelevant PCR mixture. Samples were overlaid with 100 μl light mineraloil and heated to 94° C. for 5 mins prior to thermal cycling. Reactionmixtures and thermal cycle times for this second round of PCR wereexactly as described above for selective amplification (Example 5) todetect specific mutations (i.e. primer A was paired with 2W or 2M, orprimer B was paired with 3W or 3M). Inclusion of 1 μg herring sperm DNAin each reaction was round to enhance the selectivity of the second PCR.In addition, due to the extreme sensitivity of “double” PCR, stringentprecautions were taken to avoid cross-contamination of samples and PCRreagents. Control reactions were always performed with no added DNA toensure no spurious amplification had occurred. The sequence of PCRprimer NE1 was as follows: 5′-TCATTGACAGTGCAGCT-3′ This primer annealsdownstream of primers 3W and 3M as shown in FIG. 4a.

1. A method for determining the sensitivity of an HIV-1 samples tozidovudine, which comprises: (a) isolating HIV-1 DNA extracted fromhuman cells or HIV-1 RNA isolated from body fluids, (b) hybridizing adetectably labeled oligonucleotide to the HIV-1 DNA isolated in step(a), the oligonucleotide having at its 3′ end at least 15 nucleotidescomplementary to a region of the weld type DNA sequence, itscorresponding RNA, to a region of the mutant DNA sequence set forth inFIG. 1, or its corresponding RNA, wherein the oligonucleotide terminatesof the 3′-end with said at least 15 nucleotides at the 2328, 2338, 2772,2773, or 2784 position, (c) attempting to extend the oligonucleotide atits 3′-end, (d) ascertaining the presence or absence of a detectablylabeled extended oligonucleotide, (d) correlating the presence orabsence of a detectably labeled extended oligonucleotide in step (d)with the sensitivity of the HIV-1 samples to zidovudine.
 2. A method fordetermining the sensitivity of an HIV-1 sample to zidovudine whichcomprises: (a) isolating nucleic acid from the sample, (b) hybridizing adetectably labeled oligonucleotide to the HIV-1 nucleic acid isolated instep (a), the oligonucleotide having at least 15 nucleotidescomplementary to a region of the wild type DNA sequence, itscorresponding RNA, to a region of the mutant DNA sequence set forth inFIG. 1, or its corresponding RNA, wherein said oligonucleotide having atleast 15 nucleotides contains at least one nucleotide at the 2328, 2338,2772, 2773, or 2784 position, (c) ascertaining whether or not any of theresulting hybrids of the detectably labeled oligonucleotide and nucleicacid have complementary nucleotides at one of these positions, and (d)correlating the presence of absence of a detectably labeled nucleic acidhybridization product formed instep (b) with the sensitivity of an HIV-1sample to zidovudine.
 3. In the method of claim 1 or 2, prior to step(b), the isolated nucleic acid is amplified prior to hybridization.
 4. Amethod as claimed in claim 1 or 2 wherein the detectable label on theoligonucleotide is an enzyme, radioisotope or fluorochrome.
 5. A methodfor determining the sensitivity of a patient sample containing HIV-1 tozidovudine, comprising: detecting a substitution at one or more ofpositions 2328, 2338, 2772, 2773 and/or 2784 in the reversetranscriptase gene or HIV-1 from the patient sample relative to thewildtype sequence shown in FIG. 1, wherein the presence of one or moresubstitutions correlates with decreased sensitivity of the sample tozidovudine.
 6. The method of claim 5, wherein the substitution isdetected at position
 2328. 7. The method of claim 5, wherein thesubstitution is detected at position
 2338. 8. The method of claim 5,wherein the substitution is detected at position
 2772. 9. The method ofclaim 5, wherein the substitution is detected at position
 2773. 10. Themethod of claim 5, wherein the substitution is detected at position2784.
 11. The method of claim 5, further comprising: assessing thesensitivity of the HIV sample to zidovudine from the presence or absenceof a substitution at one or more of the nucleotides.
 12. A method fordetermining the sensitivity of a patient sample containing HIV-1 tozidovudine, comprising: hybridizing an oligonucleotide to a HIV-1nucleic acid from the patient sample, wherein the oligonucleotide iscomplementary to a HIV-1 reverse transcriptase sequence or itscomplement including one or more nucleotides as positions 2328, 2338,2772, 2773, and/or 2784; detecting hybridization between theoligonucleotide and the nucleic acid to determine a nucleotide presentat one or more of positions 2328, 2338, 2773 and/or 2784, the identityof the nucleotide indicating whether the HIV sample is sensitive tozidovudine.
 13. The method of claim 12, wherein the oligonucleotide isimmobilized to a support.
 14. The method of claim 13, wherein the HIV-1nucleic acid is labelled and the oligonucleotide is unlabelled.
 15. Anoligonucleotide probe for determining a nucleotide at one or more ofpositions 2328, 2338, 2772, 2773 and/or 2784 in a HIV-1 reversetranscriptase gene, wherein the probe is complementary to a HIV-1reverse transcriptase sequence or its complement including at least oneof the positions.
 16. The oligonucleotide probe of claim 15 thathybridizes to the wildtype form of the reverse transcriptase gene shownin FIG. 1 or its complement.
 17. The oligonucldotide probe of claim 15that hybridizes to a mutant form of the reverse transcriptase gene shownin FIG. 1 or its complement.
 18. The oligonucleotide probe of claim 15,wherein the probe includes position 2328 of the HIV-1 reversetranscriptase sequence or its complement and hybridization of the probeto the sequence or its complement determines the nucleotide occupyingposition
 2328. 19. The oligonucleotide probe of claim 15, wherein theprobe includes position 2338 of the HIV-1 reverse transcriptase sequenceor its complement and hybridization of the probe to the sequence or itscomplement determines the nucleotide occupying position
 2338. 20. Theoligonucleotide probe of claim 15, wherein the probe includes position2772 of the HIV-1 reverse transcriptase sequence or its complement andhybridization of the probe to the sequence or its complement determinesthe nucleotide occupying position
 2772. 21. The oligonucleotide probe ofclaim 15, wherein the probe includes position 2773 of the HIV-1 reversetranscriptase sequence or its complement and hybridization of the probeto the sequence or its complement determines the nucleotide occupyingposition
 2773. 22. The oligonucleotide probe of claim 15, wherein theprobe includes position 2784 of the HIV-1 reverse transcriptase sequenceor its complement and hybridization of the probe to the sequence or itscomplement determines the nucleotide occupying position
 2784. 23. Amethod of screening a HIV patient being or to be treated with a drug,comprising: obtaining a sample from the patient; detecting one or moresubstitutions in a reverse transcriptase gene of a HIV genome from thepatient sample; and correlating the one or more substitutions withdecreased sensitivity to the drug.
 24. The method of claim 23, furthercomprising: diagnosing decreased sensitivity to the drug in a secondpatient infected with HIV having a reverse transcriptase gene with atleast one of the substitutions.
 25. A method for determining thesensitivity of a patient sample containing HIV- 1 to zidovudine,comprising: (a) isolating a nucleic acid sample from cells of thepatient; (b) amplifying a reverse-transcriptase sequence from thenucleic acid sample; (c) performing dideoxy sequencing of thereverse-transcriptase sequence; and (d) detecting a substitution at oneor more of positions 2328, 2338, 2772, 2773 and 2784 in the reversetranscriptase sequence of HIV- 1 from the patient sample relative to thewild type sequence shown in FIG. 1, wherein the presence of one or moresubstitutions correlates with decreased sensitivity of the sample tozidovudine.
 26. The method of claim 25, wherein step (b) is performed byamplifying HIV- 1 RNA by RT-PCR.
 27. The method of claim 25, whereinstep (b) is performed by amplifying HIV- 1 DNA by PCR.
 28. The method ofclaim 25, wherein the amplifying is performed through the use of nestedprimers.
 29. The method of claim 25, wherein the patient has beentreated with zidovudine prior to performing step (a).
 30. The method ofclaim 25, wherein the patient has not been treated with zidovudine priorto performing step (a).
 31. The method of claim 25, wherein thesubstitution is detected at position
 2328. 32. The method of claim 25,wherein the substitution is detected at position
 2338. 33. The method ofclaim 25, wherein the substitution is detected at position
 2772. 34. Themethod of claim 25, wherein the substitution is detected at position2773.
 35. The method of claim 25, wherein the substitution is detectedat position
 2784. 36. A method for determining the sensitivity of apatient sample containing HIV- 1 to zidovudine, comprising: (a)hybridizing an immobilized oligonucleotide that is at least 15nucleotides long to an HIV- 1 nucleic acid from the patient sample,wherein the oligonucleotide is complementary to an HIV- 1 reversetranscriptase sequence or its complement including one or morenucleotides at positions 2328, 2338, 2772, 2773, and/or 2754; and (b)detecting hybridization between the oligonucleotide and the nucleic acidto determine a nucleotide present at one or more of positions 2328,2338, 2772, 2773 and/or 2784, the identity of the nucleotide indicatingwhether the HIV sample is sensitive to zidovudine.
 37. The method ofclaim 36, wherein the HIV- 1 nucleic acid is labeled and theoligonucleotide is unlabeled.
 38. The method of claim 36, wherein theoligonucleotide includes position 2328 of the HIV- 1 reversetranscriptase sequence or its complement and hybridization of theoligonucleotide to the sequence or its complement determines thenucleotide occupying position
 2328. 39. The method of claim 36, whereinthe oligonucleotide includes position 2338 of the HIV- 1 reversetranscriptase sequence or its complement and hybridization of theoligonucleotide to the sequence or its complement determines thenucleotide occupying position
 2338. 40. The method of claim 36, whereinthe oligonucleotide includes position 2772 of the HIV- 1 reversetranscriptase sequence or its complement and hybridization of theoligonucleotide to the sequence or its complement determines thenucleotide occupying position
 2772. 41. The method of claim 36, whereinthe oligonucleotide includes position 2773 of the HIV- 1 reversetranscriptase sequence or its complement and hybridization of theoligonucleotide to the sequence or its complement determines thenucleotide occupying position
 2773. 42. The method of claim 36, whereinthe oligonucleotide includes position 2784 of the HIV- 1 reversetranscriptase sequence or its complement and hybridization of theoligonucleotide to the sequence or its complement determines thenucleotide occupying position
 2784. 43. The method of claim 36, whereinthe nucleic acid is isolated by amplifying HIV- 1 RNA by RT-PCR.
 44. Themethod of claim 36, wherein the nucleic acid is isolated by amplifyingHIV- 1 DNA by PCR.
 45. The method of claim 43, wherein the amplifying isperformed through the use of nested primers.
 46. The method of claim 44,wherein the amplifying is performed through the use of nested primers.47. The method of claim 36, wherein the patient has been treated withzidovudine prior to performing step (a).
 48. The method of claim 36,wherein the patient has not been treated with zidovudine prior toperforming step (a).